1. Field of the Invention
The present invention relates to systems for releasing genetic materials from a solid medium. The present invention also relates to methods for releasing genetic materials from a solid medium. The present invention further relates to methods for isolating genetic material from a biological sample.
2. Description of the Related Art
Genetic material in blood samples, tissue samples and other fluids is used for the purposes of monitoring and diagnosing genetic diseases, blood-borne parasitic diseases such as malaria, and other diseases and disorders. Genetic material further can be used for determining paternity and monitoring other unusual cell populations in blood and other fluids. Analysis of genetic material can be achieved through numerous techniques and utilizes various materials. Generally, these techniques and methods involve the initial collection of the genetic material, storage of the genetic material and then subsequent analysis of the genetic material.
Human genomic DNA is purified by a variety of methods (Sambrook and Russell (2001), Molecular Cloning, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel et al. (1992), Current Protocols in Molecular Biology, John Wiley & Sons, New York, including periodic updates). Consequently, many commercial kit manufacturers provide products for such techniques, for example: AmpReady™ (Promega, Madison, Wis.), DNeasy™ (Qiagen, Valencia, Calif.), and Split Second™ (Roche Molecular Biochemicals, Indianapolis, Ind.). These products rely on the use of specialized matrices or buffer systems for the rapid isolation of the genomic DNA molecule.
More recently, microporous filter-based techniques have surfaced as tools for the purification of genomic DNA as well as a whole multitude of nucleic acids. The advantages of filter-based matrices are that they can be fashioned into many formats that include tubes, spin tubes, sheets, and microwell plates. Microporous filter membranes as purification support matrices have other advantages within the art. They provide a compact, easy to manipulate system allowing for the capture of the desired molecule and the removal of unwanted components in a fluid phase at higher throughput and faster processing times than possible with column chromatography. This is due to the fast diffusion rates possible on filter membranes.
Nucleic acid molecules have been captured on filter membranes, generally either through simple adsorption or through a chemical reaction between complementary reactive groups present on the filter membrane or on a filter-bound ligand resulting in the formation of a covalent bond between the ligand and the desired nucleic acid.
Porous filter membrane materials used for non-covalent nucleic acid immobilization have included materials such as nylon, nitrocellulose, hydrophobic polyvinylidinefluoride (PVDF), and glass microfiber. A number of methods and reagents have also been developed to also allow the direct coupling of nucleic acids onto solid supports, such as oligonucleotides and primers (e.g., Coull et al. (1986), Tetrahedron Lett 27:3991-3994; Connolly (1987), Nucleic Acids Res 15:3131-3139, 1987; Connolly and Rider (1985), Nucleic Acids Res 12:4485-4502; Yang et al. (1998), Proc Natl Acad Sci USA 95:5462-5467). UV cross-linking of DNA (Church et al. (1984), Proc Natl Acad Sci USA 81:1991-1995), RNA (Khandjian et al. (1986), Anal Biochem 159:227-232) to nylon membranes, The Generation Capture Column Kit (Qiagen, Valencia, Calif.) QIAamp DNA Blood Mini Kit, QIAamp DNA Mini Kit (Qiagen, Valencia, Calif.), ChargeSwitch® technology (Invitrogen, Corp., Carlsbad, Calif.), MagaZorb® isolation kits (Cortex Biochem, Inc., San Leandro, Calif.) and NucliSENS® Isolation Kit (bioMérieux, Inc., Durham, N.H.) have also been reported.
Many chemical methods have been utilized for the immobilization of molecules such as nucleic acids on filter membranes. For example, activated paper (TransBind™, Schleicher & Schuell Ltd., Keene, N.H.), carbodimidazole-activated hydrogel-coated PVDF membrane (Immobilin-IAV™, Millipore Corp., Bedford, Mass.), MAP paper (Amersham, Littlechalfont Bucks, Wis.), activated nylon (BioDyne™, Pall Corp., (Glen Cove, N.Y.), DVS- and cyanogen bromide-activated nitrocellulose. Membranes bound with specific ligands are also known such as the SAM2™ Biotin Capture Membrane (Promega) which binds biotinylated molecules based on their affinity to streptavidin or MAC affinity membrane system (protein A/G) (Amicon, Bedford, Mass.). A primary disadvantage of covalent attachment of biomolecules onto activated membranes is that the covalently bound molecules can not be retrieved from the filter membrane.
More recently, glass microfiber has been shown to specifically bind nucleic acids from a variety of nucleic acid containing sources very effectively (e.g., Itoh et al. (1997), Nucleic Acids Res 25:1315-1316; Andersson et al (1996), BioTechniques 20:1022-1027; U.S. Pat. No. 5,910,246). Under the correct salt and buffering conditions, nucleic acids will bind to glass or silica with high specificity. U.S. Pat. No. 5,234,809 describes a method in which nucleic acids are bound to a solid medium in the form of silica particles, in the presence of a chaotropic agent such as a guanidinium salt, and thereby separated from the remainder of the sample. International published application No. WO 91/12079 describes a method whereby nucleic acid is trapped on the surface of a solid medium by precipitation. Generally speaking, alcohols and salts are used as precipitants. U.S. Pat. No. 6,617,105 describes a method for isolating nucleic acids from cells in which cells are non-specifically or specifically bound to a solid medium, such as glass, silica, latex or polymeric materials, the cells are lysed allowing the DNA to be bound to the same solid phase which is then recovered. A similar process using a porous matrix is described in U.S. Pat. No. 5,653,141.
Nucleic acids or genetic material can be immobilized to a cellulosic-based dry solid support or filter (FTA® filter; FTA® cellulosic filter material; Whatman, plc). See, for example, U.S. Pat. Nos. 5,496,562, 5,756,126, 5,807,527, 6,322,983 and 6,627,226. The solid support described is conditioned with a chemical composition that is capable of carrying out several functions: (i) lyse intact cellular material upon contact, releasing genetic material, (ii) enable and allow for the conditions that facilitate genetic material immobilization to the solid support (probably by a combination of mechanical and chaotrophic), (iii) maintain the immobilized genetic material in a stable state without damage due to degradation, endonuclease activity, UV interference, and microbial attack, and (iv) maintain the genetic material as a support-bound molecule that is not removed from the solid support during any down stream processing (e.g., Del Rio et al. (1995), BioTechniques 20:970-974).
The usefulness of the so called FTA® cellulosic filter material described in the above patents has been illustrated for several nucleic acid techniques such as bacterial ribotyping (Rogers and Burgoyne (1997), Anal Biochem 247: 223-227), detection of single base differences in viral and human DNA (Ibrahim et al. (1998), Anal Chem 70: 2013-2017), DNA databasing (Ledray et al. (1997), J Emergency Nursing 23:156-158), automated processing for STR electrophoresis (Belgrader and Marino (1996), L.R.A. 9:3-7; Belgrader et al. (1995), BioTechniques 19:427-432), and oligonucleotide ligation assay for diagnostics (Baron et al. (1996), Nature Biotech 14:1279-1282).
As illustrated above, various materials and solid media have been and continue to be utilized to provide a base for performing any desired analysis of the genetic material. Those materials include, for example, FTA® filter paper or FTA®-coated materials. In particular, FTA®-coated materials have been successfully utilized for preparing all types of genetic material for subsequent genetic analysis. Genetic material prepared using FTA®-coated materials and FTA® techniques yields highly purified material bound to the cellulosic base filter for the duration of various subsequent applications and amplification reactions. FTA®-coated base filter materials include, but are not limited to Whatman cellulosic BFC-180, 31-ET, glass microfibers, and other similar filter materials known to those of skill in the art.
It is known that high molecular weight genetic material does not release well from any media. For example, it has been shown that nucleic acid or genetic material applied to, and immobilized to, FTA® filters cannot be simply removed, or eluted from the solid support once bound (Del Rio et al. (1995), BioTechniques 20:970-974). This is a major disadvantage for applications where several downstream processes are required from the same sample, such a STR profiling and genotyping. This disadvantage has recently been confirmed. Specifically, it has been shown that not all commercial methods are capable of extracting sufficient DNA for use in a whole genome amplification step prior to a quantitative PCR (Q-PCR) reaction (Sjöholm et al. (2007), Clin Chem 53:1401-1407). These commercial methods are extremely cumbersome and many hours are required to obtain enough material for use in a Q-PCR reaction.
The difficulty in removing genetic material from FTA® filters has been well recognized in the art, and several techniques have been developed for removing genetic material from FTA® filters. One technique includes the use of chemical methods, such as the use of special buffer compositions (U.S. Pat. No. 6,410,725). This technique, as well as other techniques that rely on the use of chemical methods to release the genetic material, require additional reagents and steps, thus increasing the complexity of the isolation of genetic material. Other techniques include photolysis (U.S. Pat. No. 6,972,329), heat (U.S. Pat. No. 6,645,717) and treatment of the genetic material on the paper for detection (U.S. Pat. No. 6,746,841).
Although the above methods speed up the nucleic acid separation process, a need still exists for methods which are quick and simple to perform, which have higher efficiency, and in particular which are readily amenable to isolating nucleic acids from cells for use in microfluidic environments, such as microfluidic PCR methods.